腐败梭菌 α 毒素突变体可溶性表达及其对小鼠的免疫评价

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中图分类号：S852.616.3 文献标志码：A 文章编号：0366-6964（2025）10-5148-11

Abstract： This study aims to obtain soluble and highly immunogenic mutants of Clostridium septicum alpha toxin. Site mutations in the alpha toxin protein were predicted using the MPEPE prediction tool. Expression vectors were constructed and expressed in E .coli for validation. The solubility， toxicity，and immunogenicity in mice of various rCsa mutants were compared. The results showed that the rCsa mutants were correctly expressed in E . coli，with all bands appearing at approximately . The soluble expression levels of rCsaS398D ， rCsaK405M ，and rCsaN397D accounted for 9.5% ， 9.4% ，and 10.5% of total expressed proteins in the bacterial lysate supernatant，respectively. The ratios of protein amounts in the supernatant to those in the pellet were 2.5，2.6，and 2.9，respectively. rCsaN397D showed the highest soluble expression level among the three rCsa mutants，and it was also superior to the 40% solubility of rCsa observed in previous experiments. Mouse toxicity tests indicated that none of the three rCsa mutants lost their toxicity. Vero cell toxicity tests showed that concentrations as low as 0.0016μg⋅mL-1 of each rCsa mutant could induce significant cytopathic effects （CPE）. ELISA detection of IgG antibody levels in mouse serum 2l days after the second immunization revealed that the S/N values of the rCsaN397D group serum were significantly higher than those of the rCsaS398D and rC（204号 saK405M groups ⋅P<0. 01 ）.Neutralizing titers of mouse serum from the second immunization tested on Vero cells showed that the neutralizing titer of rCsaN397D was 6400 MLD ⋅mL-1 ， which was significantly higher than those of the rCsaS398D and rCsaK405M groups（ P< 0.0001），and also higher than the neutralizing titer of unmutated rCsa from previous experiments. In summary，rCsaN397D outperformed the other two rCsa mutants in terms of solubility and immunogenicity，and its immunogenicity was better than that of the unmutated rCsa. Therefore，rCsaN397D can be considered a candidate antigen for a genetically engineered subunit vaccineagainst Clostridium septicum.

Keywords： Clostridium se pticumα toxin; MPEPE;virulence; solubility;immunogenicity ∗ Corresponding authors： FAN Xuezheng， E-mail：fanxuezheng@caas. cn; WEI Yingyi， E-mail： weiyingyi@gxu. edu. cn

腐败梭菌（CLostridiumsepticum，CS）可引起多种动物的恶性水肿、肌肉坏死、气性坏疽以及坏死性肠炎等疾病，还可引起羊快疫，此病发病快、病程短、治疗难度极大，给养羊业造成了巨大经济损失[1]。（剩余18318字）